Modern molecular biology requires the isolation of nucleic acids from a variety of sources comprising complex nucleic acids mixture with other compounds such as proteins, lipids and other cellular constituents. Particularly important examples of such mixtures include soluble nucleoproteins, which consist of nucleic acid molecules of varying length, complexed with proteins, e.g., histones. These complexes can be released into the bloodstream as a result of the normal apoptosis or other forms of cell death. It has been shown that eventually they cross the kidney barrier (Tr-DNA/RNA) and can be detected in the urine (Umansky, S. R., et al. 1982, Biochim. Biophys. Acta 655:9-17; Lichstenstein, A. V., et al. 2001, Ann NY Acad Sci, 945:239-249; Umansky, S & Tomei, D. 2006 Expert Rev. Mol. Diagn. 6:153-163). Because they are soluble, and released from cells throughout the body they can be useful as indicators for the detection and monitoring of diseases and abnormal conditions which may be present in areas of the body other than where the fluid is obtained (Al-Yatama et al. 2001, Prenat Diagn, 21:399-402, Utting, M., et al. (2002), Clin Cancer Res, 8:35-40). Most of currently existing technologies can be designed for isolation of high molecular weight cellular DNA and RNA. These methods require several steps. First, cells must be lysed. Second, nuclease activity has to be inhibited to prevent degradation of the released nucleic acids. Third, the nucleic acids should be separated from proteins. This deproteinization step may include extraction with a variety of organic solvents, including phenol and/or chloroform, followed by precipitation with ethanol, (Maniatis et al., “Molecular Cloning. A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Another method for nucleic acids extraction can be based on their adsorption on silica from highly concentrated solutions of chaotropic agents with subsequent elution at a low ionic strength. These conventional methods suffer from the disadvantages of being time-consuming and laborious, and they can also involve the use of chemical reagents that are hazardous and/or toxic to the worker and/or the environment.
Purification of DNA and/or RNA from cell free nucleoprotein complexes may not require the cell lysis step. However, there is a large body of evidence showing that the cell free circulating nucleic acids in bodily fluids can be present in very low concentrations and are fragmented to the average size of 150-300 bp, which introduces additional requirements to a purification procedure. First, for a large specimen volume it can be technically difficult to perform phenol deproteinization, automation of the process is almost impossible, and use of extremely toxic phenol in clinical lab setting is impractical. Silica-based methods may be much more suitable for isolation of nucleic acids from diluted solutions. However, first, significant additional dilution of original nucleic acid solution can be necessary to get high enough concentration of a chaotropic agent, and DNA isolation from large volumes becomes a problem. Second, binding of single stranded and short nucleic acid fragments (less than 200 bp) to silica is not very effective, and they can be lost during purification.
Anion exchange media has also been used for the fractionation and isolation of nucleic acids, although the biological sample containing them is usually first been processed to free the nucleic acids from other cellular components (Yamakawa et al., Analytical Biochemistry, 24:242-250 (1996).
There remains an unmet need in the art for a novel method of isolating and preserving cell-free nucleic acids from body fluids wherein such method avoids multiple proceeding to free the nucleic acids from cellular material, provides nucleic acid sequences of various lengths, preserves the integrity of nucleic acids and is readily adaptable to high-throughput of samples for the diagnostic analysis.